This application is the National Phase application of International Application No. PCT/IB98/00706, filed Mar. 2, 1998.
This invention relates to apoptosis.
Apoptosis, which is also referred to as programmed cell death, is a form of cell death characterized by membrane blebbing and nuclear DNA fragmentation. Apoptotic cell death is morphologically distinct from necrotic cell death and is important in the normal development and maintenance of multicellular organisms. Dysregulation of apoptosis has been implicated in a number of human diseases. An inappropriate suppression of apoptosis in a cell may lead to the uncontrolled propagation of that cell. Such an event would favor, for example, the development of cancer. In contrast, a failure to control the extent of apoptotic cell death may lead to degeneration of specific tissues and cell-types. For example, an inappropriately high level of apoptosis in leukocytes may result in acquired immunodeficiency. Likewise, certain neurodegenerative disorders may result from an inappropriately high level of apoptosis in neuronal cells and tissues.
Although apoptotic cell death is initially triggered by a specific death signal received, for example, by ligation of the Fas cell surface molecule, execution of the apoptotic pathway occurs only upon the activation of members of the Ced-3/ICE (caspase) family of cysteine proteases. There are at least 10 known members of the caspase family whose activities lead to site-specific cleavage and consequent activation/inactivation of various target molecules. FLICE and related caspases may initiate apoptosis by activating a downstream caspase cascade, including CPP32 (caspase-3).
The decision to engage the apoptotic execution pathway in response to specific death signals depends on the status of various cellular regulators of apoptosis, including p53 and the Bcl-2/Bax set point. The latter set point arises through heterodimerization between the Bcl-2/Bcl-XL family of suppressors and promoters, respectively, in which the ratio of the heterodimerizing partners determines the outcomexe2x80x94cell death or cell survivalxe2x80x94in response to various death signals. Bad, a more distantly related family member, is a direct regulator of the set point, by a mechanism that is governed by phosphorylation. The phosphorylation may, in turn, be affected by Bcl-2-dependent recruitment of Raf-1 kinase.
Although it is now known that Bcl-2/Bcl-XL controls the apoptotic execution pathway at a point that is either at or upstream of pro-enzyme activation of the caspases, how this is achieved remains to be elucidated. There thus remains a need to identify Bcl-2 binding proteins that are functionally linked to apoptosis. There also remains a need to identify factors that interact with Bcl-2 and modulate the apoptotic signalling pathway. Further, it would be useful to identify factors which enable a signalling of the apoptotic pathway from Bcl-2 and interacting factors to the caspases involved therein.
We have discovered that p28 Bap31 polypeptides, nucleic acids, and antibodies may be used for the detection and treatment of conditions involving apoptosis and for the identification of therapeutic molecules.
In a first aspect, the invention features a substantially pure p28 Bap31 polypeptide fragment that modulates apoptosis.
In a second aspect, the invention features a substantially purified nucleic acid molecule encoding a substantially pure p28 Bap31 polypeptide fragment that modulates apoptosis.
In various embodiments of the first two aspects of the invention, the fragment includes a domain that is required for an association of p28 Bap31 with pro-FLICE, or a domain that is required for an association of p28 Bap31 with a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL). In other preferred embodiments, the fragment either increases apoptosis or inhibits apoptosis. In yet another embodiment of the first and second aspects of the invention, the fragment is from a mammal (e.g., a human or a mouse).
In an third aspect, the invention features a substantially pure polypeptide that modulates apoptosis, the polypeptide having 50% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. In preferred embodiments, the polypeptide has 70% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, or has 80% or greater amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.
In a fourth aspect, the invention features a method for modulating apoptosis in a cell that includes administering to the cell a compound that alters p28 Bap31 biological activity, the compound being administered at a dosage which is sufficient to modulate the p28 Bap31 biological activity. In various preferred embodiments of this aspect of the invention, the p28 Bap31 biological activity may be cleavage of the p28 Bap31 polypeptide to produce a p20 product, formation of a complex of the p28 Bap31 polypeptide with pro-FLICE, formation of a complex of the p28 Bap31 polypeptide with a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL), binding of the p28 Bap31 polypeptide by an antibody that specifically binds to p28 Bap31, or expression of the p28 Bap31 polypeptide in the cell. In various other embodiments, the cell is from a mammal (e.g., a human or a rodent).
In another embodiment of the fourth aspect of the invention, the modulating is inhibiting. In another embodiment, where the modulating is inhibiting, the cell is in a mammal with a degenerative disease (e.g., a neurodegenerative disease, cirrhosis of the liver, a myelodysplastic syndrome, an ischemic injury, an infection with HIV, or a bone degenerative disease). In yet another embodiment, where the-modulating is inhibiting, the compound may be a p28 Bap31 antisense nucleic acid molecule, an antibody that specifically binds to p28 Bap31 (e.g., a p28 Bap31 neutralizing antibody), a p20 antisense nucleic acid molecule, an antibody that specifically binds to p20 (e.g., a p20 neutralizing antibody), a p20-inhibiting amount of a Bcl-2 protein, or a p20-inhibiting amount of a nucleic acid molecule encoding a Bcl-2 polypeptide, where the nucleic acid molecules are positioned for expression in said cell.
In another embodiment of the fourth aspect of the invention, the modulating is increasing. In another embodiment, where the modulating is increasing, the cell is in a mammal with a neoplasia. In another embodiment, where the modulating is increasing, the compound may be a p28 Bap31 polypeptide, a p20 product that is a cleavage product of p28 Bap31, or a nucleic acid molecule encoding a p28 Bap31 polypeptide, where the nucleic acid molecule is positioned for expression in the cell.
In a fifth aspect, the invention features a method for detecting a compound that modulates apoptosis that includes the steps of: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, the first fusion protein including a polypeptide fragment of p28 Bap31 covalently bonded to a binding moiety, the binding moiety capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene capable of expressing a second fusion protein, the second fusion protein including a polypeptide fragment of a second protein covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, a change in the reporter gene expression identifying a compound that modulates apoptosis. In one embodiment of this aspect, the cell is a yeast cell.
In a sixth aspect, the invention features a method for detecting a compound that modulates apoptosis that includes the steps of: (a) providing a cell having: (i) a reporter gene operably linked to a DNA-binding-protein recognition site; (ii) a first fusion gene capable of expressing a first fusion protein, the first fusion protein including a polypeptide fragment of a second protein covalently bonded to a binding moiety, the binding moiety capable of specifically binding to the DNA-binding-protein recognition site; and (iii) a second fusion gene capable of expressing a second fusion protein, the second fusion protein including a polypeptide fragment of p28 Bap31 covalently bonded to a gene activating moiety; (b) exposing the cell to the compound; and (c) measuring reporter gene expression in the cell, a change in the reporter gene expression identifying a compound that modulates apoptosis. In one embodiment of this aspect, the cell is a yeast cell.
In a seventh aspect, the invention features a method for identifying a compound that modulates apoptosis that includes the steps of: (a) providing a first polypeptide including a region of p28 Bap31, the region of p28 Bap31 including a first domain that interacts with a second protein; (b) allowing an interaction of the first polypeptide with a second polypeptide that includes a region of the second protein, the region of the second protein including a second domain that interacts with the p28 Bap31; (c) contacting the interaction of the first polypeptide and the second polypeptide with a candidate compound; and (d) measuring the interaction of the first polypeptide and the second polypeptide, a change in the interaction of the first polypeptide and the second polypeptide in the presence of the candidate compound relative to an interaction of the first polypeptide and the second polypeptide not contacted with the candidate compound identifying the presence of a compound that modulates apoptosis.
In various embodiments of the fifth, sixth, and seventh aspects of the invention, the second protein is a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL).
In an eighth aspect, the invention features a method for identifying a compound that modulates apoptosis that includes: (a) providing a cell expressing p28 Bap31 polypeptide; and (b) contacting the cell with a candidate compound and monitoring the level of p28 Bap31 biological activity, a change in the level of p28 Bap31 biological activity in response to the candidate compound relative to a level of p28 Bap31 biological activity in a cell not contacted with the candidate compound identifying a compound that modulates apoptosis.
In various preferred embodiments of the eighth aspect of the invention, the p28 biological activity may be cleavage of the p28 Bap31 polypeptide to produce a p20 product, formation of a complex of the p28 Bap31 polypeptide with pro-FLICE, formation of a complex of the p28 Bap31 polypeptide with a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL), binding of the p28 Bap31 polypeptide by an antibody that specifically binds to p28 Bap31, or expression of the p28 Bap31 polypeptide in the cell. In various other embodiments, the cell is from a mammal (e.g., a human or a rodent).
In another embodiment of the fifth, sixth, seventh, and eighth aspects of the invention, where the change is a decrease, the compound is useful for inhibiting apoptosis. In another embodiment, where the change is a decrease, the compound may be used to treat an animal with a degenerative disease (e.g., a neurodegenerative disease, cirrhosis of the liver, a myelodysplastic syndrome, an ischemic injury, an infection with HIV, or a bone degenerative disease). In yet another embodiment, where the change is an increase, the compound is useful for increasing apoptosis. In yet another embodiment, where the change is an increase, the compound may be used to treat an animal with neoplasia (e.g., a cancer, a hyperplastic disorder, or a benign tumor).
In a ninth aspect, the invention features a method for diagnosing a mammal for the presence of a disease involving altered apoptosis or an increased likelihood of developing the disease, where the method includes measuring the level of p28 Bap31 biological activity in a sample from the mammal, a change in the level of p28 Bap31 biological activity in the sample relative to a level of p28 Bap31 biological activity in a sample from an unaffected mammal being an indication that the mammal has the disease or increased likelihood of developing the disease.
In various preferred embodiments of the ninth aspect of the invention, the p28 biological activity may be cleavage of the p28 Bap31 polypeptide to produce a p20 product, formation of a complex of the p28 Bap31 polypeptide with pro-FLICE, formation of a complex of the p28 Bap31 polypeptide with a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL), binding of the p28 Bap31 polypeptide by an antibody that specifically binds to p28 Bap31, or expression of the p28 Bap31 polypeptide in the cell. In another embodiment, the mammal is a human or a rodent.
In another embodiment of the ninth aspect of the invention, where the change is a reduction indicates that the mammal has the disease or increased likelihood of developing the disease, wherein the disease is caused by decreased apoptosis. In another embodiment, where the change is a reduction, the disease may be neoplasia (e.g., a cancer, a hyperplastic disorder, or a benign tumor).
In yet another embodiment of the ninth aspect of the invention, where the change is an increase indicates that the mammal has the disease or increased likelihood of developing the disease, wherein the disease is caused by increased apoptosis. In another embodiment, where the change is an increase, the disease may be a degenerative disease (e.g., a neurodegenerative disease, cirrhosis of the liver, a myelodysplastic syndrome, an ischemic injury, an infection with HIV, or a bone degenerative disease).
In a tenth aspect, the invention features a method for identifying a nucleic acid molecule encoding a p28 Bap31 polypeptide that includes: (a) providing a cell; (b) introducing by transformation into the cell a candidate nucleic acid molecule, the nucleic acid molecule being positioned for expression in the cell; and (c) determining whether the transformed cell exhibits an increased level of apoptosis relative to a cell not transformed with the candidate nucleic acid molecule, wherein the increased level of apoptosis in the transformed cell identifies a nucleic acid molecule encoding a p28 Bap31 polypeptide.
In an eleventh aspect, the invention features a method for identifying a nucleic acid molecule encoding a p28 Bap31 polypeptide that includes: (a) providing a cell; (b) introducing by transformation into the cell a candidate nucleic acid molecule, the nucleic acid molecule being positioned for expression in the cell; and (c) determining whether the transformed cell exhibits an increased level of p28 Bap31 biological activity relative to a cell not transformed with the candidate nucleic acid molecule, wherein the increased level of p28 Bap31 biological activity in the transformed cell identifies a nucleic acid molecule encoding a p28 Bap31 polypeptide.
In a twelfth aspect, the invention features a method for identifying a nucleic acid molecule encoding a protease that cleaves p28 Bap31 into p20 that includes: (a) providing a cell expressing a p28 Bap31 polypeptide; (b) introducing by transformation into the cell a candidate nucleic acid molecule, the nucleic acid molecule being positioned for expression in the cell; and (c) determining whether the transformed cell exhibits an increased level of p20 expression relative to a cell not transformed with the candidate nucleic acid molecule, wherein the increased level of p20 expression in the transformed cell identifies a nucleic acid molecule encoding a protease that cleaves p28 Bap31 into p20.
In a thirteenth aspect, the invention features a method for identifying a nucleic acid molecule encoding a protease that cleaves p28 Bap31 into p20 that includes: (a) providing a cell expressing a p28 Bap31 polypeptide; (b) introducing by transformation into the cell a candidate nucleic acid molecule, the nucleic acid molecule being positioned for expression in the cell; and (c) determining whether the transformed cell exhibits an increased level of apoptosis relative to a cell not transformed with the candidate nucleic acid molecule, wherein the increased level of apoptosis in the transformed cell identifies a nucleic acid molecule encoding a protease that cleaves p28 Bap31 into p20.
In a preferred embodiment of the tenth, eleventh, twelfth, and thirteenth aspects of the invention, the cell is from a mammal (e.g., a human or a rodent).
In a fourteenth aspect, the invention features a kit for diagnosing a mammal for the presence of a disease involving altered apoptosis or an increased likelihood of developing the disease, where the kit includes a substantially pure antibody that specifically binds a p28 Bap31 polypeptide, where the antibody modulates apoptosis. In one embodiment of this aspect of the invention, the kit further includes a means for detecting the binding of the antibody to the p28 Bap31 polypeptide.
In a fifteenth aspect, the invention features a substantially pure antibody that specifically binds to the p28 Bap31 polypeptide, where the antibody modulates apoptosis. In preferred embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, or a neutralizing antibody.
In a sixteenth aspect, the invention features a transgenic cell (e.g., an embryonal cell) that has a knockout mutation of an endogenous p28 Bap31 gene. In one embodiment, the mutation includes an insertion of exogenous DNA.
In a seventeenth aspect, the invention features a transgenic animal generated from a transgenic cell (e.g., an embryonal cell) that has a knockout mutation of an endogenous p28 Bap31 gene, where the endogenous p28 Bap31 gene is not expressed in the transgenic animal. In one embodiment of this aspect, the germ-line cells and somatic cells of the transgenic animal do not express the endogenous p28 Bap31 gene. In another embodiment, the transgenic animal includes germ-line cells and somatic cells expressing a nucleic acid molecule encoding a truncated or a mutated p28 Bap31 polypeptide.
In an eighteenth aspect, the invention features a vector including a knockout mutation in a DNA sequence encoding a p28 Bap31 gene, where the gene includes: (a) a first region corresponding to a 5xe2x80x2 sequence of the p28 Bap31 gene, wherein the initiator methionine codon of the p28 Bap31 gene is absent from the sequence; (b) a second region including DNA including a drug-resistance cassette, the DNA capable of conferring resistance to the drug in a cell when the DNA is present in the cell; and (c) a third region corresponding to a 3xe2x80x2 sequence of the p28 Bap31 gene.
In a nineteenth aspect, the invention features a vector including a knockout mutation in a DNA sequence encoding a p28 Bap31 gene, where the gene includes: (a) a first region corresponding to a 5xe2x80x2 sequence of the p28 Bap31 gene, wherein the initiator methionine codon of the p28 Bap31 gene is absent from the sequence; (b) a second region including DNA including a drug-resistance cassette, the DNA capable of conferring resistance to the drug in a cell when the DNA is present in the cell; and (c) a third region corresponding to a 3xe2x80x2 sequence of the p28 Bap31 gene.
In a twentieth aspect, the invention features a method of producing a transgenic mammal lacking expression of an endogenous p28 Bap31 polypeptide that includes: (a) introducing by homologous recombination a nucleic acid molecule encoding a knockout mutation of a p28 Bap31 gene into a locus occupied by an endogenous p28 Bap31 gene, the locus present in the genome of an embryonal cell of the mammal; and (b) growing the embryonal cell to produce the transgenic mammal.
In a twenty-first aspect, the invention features a therapeutic composition that includes, as an active ingredient, a p20 cleavage product of p28 Bap31, the active ingredient being formulated in a physiologically acceptable carrier, where the composition modulates apoptosis.
In a twenty-second aspect, the invention features a therapeutic composition that includes, as an active ingredient, a p28 Bap31 polypeptide, the active ingredient being formulated in a physiologically acceptable carrier, where the composition modulates apoptosis.
In a twenty-third aspect, the invention features a therapeutic composition that includes, as an active ingredient, an antibody that specifically binds p28 Bap31, the active ingredient being formulated in a physiologically acceptable carrier, where the composition modulates apoptosis.
In a twenty-fourth aspect, the invention features a therapeutic composition that includes, as an active ingredient, an antisense nucleic acid molecule that corresponds to p28 Bap31, the active ingredient being formulated in a physiologically acceptable carrier, where the composition modulates apoptosis.
In a twenty-fifth aspect, the invention features the use of a p28 Bap31 polypeptide for the manufacture of a medicament for the modulation of apoptosis.
In a twenty-sixth aspect, the invention features the use of an antibody that specifically binds to p28 Bap31 for the manufacture of a medicament for the modulation of apoptosis.
In a twenty-seventh aspect, the invention features the use of an antisense nucleic acid molecule corresponding to p28 Bap31 for the manufacture of a medicament for the modulation of apoptosis.
In a twenty-eighth aspect, the invention features the use of a p20 cleavage product of p28 Bap31 for the manufacture of a medicament for the modulation of apoptosis.
In a twenty-ninth aspect, the invention features the use of an antibody that specifically binds to a p20 cleavage product of p28 Bap31 for the manufacture of a medicament for the modulation of apoptosis.
In a thirtieth aspect, the invention features the use of an antisense nucleic acid molecule corresponding to a p20 cleavage product of p28 Bap31 for the manufacture of a medicament for the modulation of apoptosis.
As summarized above, a p28 Bap31 nucleic acid molecule, polypeptide, or antibody may be used to modulate apoptosis. Furthermore, a p28 Bap31 nucleic acid molecule, polypeptide, or antibody may be used in the discovery and/or manufacture of a medicament for the modulation of apoptosis.
By xe2x80x9cp28 Bap31,xe2x80x9d xe2x80x9cp28 Bap31 protein,xe2x80x9d or xe2x80x9cp28 Bap31 polypeptidexe2x80x9d is meant a protein, or a polypeptide fragment thereof, that can interact with a Bcl-2 protein (including, without limitation, Bcl-2 and Bcl-XL) or a pro-FLICE protein, that can participate in apoptosis, or that can be cleaved to produce a p20 product (i.e., an approximately 20 kDa product), that can induce apoptosis when expressed in a cell. Preferably, a p28 Bap31 protein has an amino acid sequence that is at least 50% identical to the amino acid sequence of human Bap31 (GenBank accession number X81817) or to the amino acid sequence of human CDM (GenBank accession number Z31696), more preferably at least 60% identical, more preferably at least 70% identical, still more preferably at least 80% identical, and most preferably at least 90% identical to at least one of these sequences. It will be understood that a fragment thereof has a p28 Bap31 biological activity that is observed with full length p28 Bap31 protein; preferably, the p28 Bap31 biological activity is at least 50% as observed with fill length p28 Bap31.
Polypeptide fragments of p28 Bap31 that are a part of the invention include those fragments that bind Bcl-2 polypeptides, those fragments that are capable of selecting an antibody which specifically binds p28 Bap31, and those fragments that can modulate apoptosis in a cell.
By xe2x80x9cp28 Bap31 genexe2x80x9d is meant a gene encoding p28 Bap31. Preferably, sequences include sequences which encode polypeptide fragments of the p28 Bap31, as defined above. In preferred embodiments, included as a part of the gene are the nucleic acid sequences flanking the 5xe2x80x2 and 3xe2x80x2 regions of the coding region of the p28 Bap31 gene sequence. Mammalian p28 Bap31 genes include nucleotide sequences isolated from any mammalian source. Preferably, the mammal is a human.
By xe2x80x9cpurified antibodyxe2x80x9d is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., a p28 Bap31 specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced polypeptide or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a polypeptide but that does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, that naturally includes protein. One preferred antibody specifically binds to the p28 Bap31 polypeptide.
By xe2x80x9cp28 Bap31 biological activity,xe2x80x9d as used herein in reference to p28 Bap31, is meant any one of the biological activities of a p28 Bap31 protein (or a polypeptide fragment thereof). P28 Bap31 biological activities include, without limitation, an ability of p28 Bap31 to be cleaved to produce a p20 product, an ability to form a complex with a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL), an ability to form a complex with pro-FLICE, an ability to be bound by an antibody that specifically binds p28 Bap31, an ability to be expressed, and an ability to participate in apoptosis.
By a xe2x80x9ccompound that alters p28 Bap31 biological activity,xe2x80x9d is meant a compound that increases or reduces any biological activity of p28 Bap31 (or a fragment thereof), as defined above. Such a compound may be, without limitation, a compound that increases the transcription of p28 Bap31, a compound that increases p28 Bap31 protein expression levels, a p28 Bap31 antisense nucleic acid molecule, an antibody that specifically binds to p28 Bap31, a p20 antisense nucleic acid molecule, an antibody that specifically binds to p20, a p20-inhibiting amount of a Bcl-2 protein, a p20-inhibiting amount of a Bcl-2-expressing nucleic acid molecule, a p28 Bap31 protein (or fragment thereof), a p20 product (or fragment thereof), and a nucleic acid molecule encoding a p28 Bap31 polypeptide (or fragment thereof).
By a xe2x80x9cneutralizing antibody,xe2x80x9d as used herein in reference to an antibody that specifically binds p28 Bap31, is meant an antibody that interferes with any of the biological activities of the p28 Bap31 protein. For example, a p28 Bap31 neutralizing antibody may interfere with the ability of p28 Bap31 to participate in apoptosis, for example, by inhibiting cleavage of p28 Bap31 to produce the p20 product. A p28 Bap31 neutralizing antibody may also interfered with the ability of p28 Bap31 to form a complex with a Bcl-2 protein (e.g., Bcl-2 or Bcl-XL) or to form a complex with pro-FLICE. Such p28 Bap31 neutralizing antibodies may reduce the ability of p28 Bap31 and polypeptide fragments thereof to participate in apoptosis and to associate with pro-FLICE and/or a Bcl-2 protein by, preferably 50%, more preferably by 70%, and most preferably by 90% or more. Standard assays of apoptosis and protein:protein interactions, including those described herein, may be used to assess potentially neutralizing p28 Bap31 antibodies.
By xe2x80x9cmodulating apoptosisxe2x80x9d or xe2x80x9caltering apoptosisxe2x80x9d is meant increasing or decreasing the number of cells that would otherwise undergo apoptosis in a given cell population. Preferably, the cell population is selected from a group including T cells, neuronal cells, fibroblasts, myocardial cells, or any other cell line known to undergo apoptosis in a laboratory setting, for example, human epithelial KB cells infected with adenovirus type 5 lacking expression of E1B 19K (pm1716/2072). It will be appreciated that the degree of modulation provided by p28 Bap31 or p28 Bap31 modulating compounds in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis which identifies a p28 Bap31 or a compound which modulates this proteins.
By xe2x80x9cincreasing apoptosisxe2x80x9d is meant any increase in the number of cells that undergo apoptosis relative to an untreated control. Preferably, the increase is at least 25%, more preferably the increase is at least 50%, and most preferably the increase is at least one-fold.
By xe2x80x9cinhibiting apoptosisxe2x80x9d is meant any decrease in the number of cells that undergo apoptosis relative to an untreated control. Preferably, the decrease is at least 25%, more preferably the decrease is at least 50%, and most preferably the decrease is at least one-fold.
By xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% identity to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine; threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the polypeptide is a p28 Bap31 polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure p28 Bap31 polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding a p28 Bap31 polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A polypeptide is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those which naturally occur in eukaryotic organisms but are synthesized in E. coli or other prokaryotes, or are synthesized in viruses.
By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic nucleic acid of a prokaryote or a eukaryote cell; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cembryonal cellxe2x80x9d is meant a cell that is capable of being a progenitor to all the somatic and germ-line cells of an organism. Embryonal cells are also referred to as embryonic stem cells, or ES cells. Preferably, the embryonal cells of the invention are mammalian embryonal cells.
By xe2x80x9cendogenous,xe2x80x9d as used herein in reference to a gene or a polypeptide, is meant a gene or polypeptide that is normally present in an organism.
By xe2x80x9cgerm-line cellxe2x80x9d is meant a cell, progenitor, or progeny thereof, which is a product of a meiotic cell division.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a nucleic acid molecule encoding (as used herein) a p28 Bap31 polypeptide.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules into a cell. Lipofection, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the teachings which may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven micro projectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts which include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., mitochondria), bacteria, yeast, animal tissue, and cultured cells.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of nucleic acid which is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from that cell. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell which includes a nucleic acid sequence which is inserted by artifice into a cell and becomes part of the genome of the transgenic organism which develops from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal. Although transgenic mice represent a preferred embodiment of the invention, other transgenic mammals including, without limitation, transgenic rodents (for example, hamsters, guinea pigs, rabbits, and rats), and transgenic pigs, cattle, sheep, and goats may be constructed by standard techniques and are included in the invention. Preferably, the transgene is inserted by artifice into the nuclear genome.
By xe2x80x9cknockout mutationxe2x80x9d is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% relative to the unmutated gene. The mutation may, without limitation, be an insertion, deletion, frameshift mutation, or a mis-sense mutation. Preferably, the mutation is an insertion or deletion (e.g., the p28 Bap31-NEO knockout gene described herein), or is a frameshift mutation that creates a stop codon.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the nucleic acid molecule is operably linked to a nucleic acid sequence which directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., a p28 Bap31 polypeptide, recombinant polypeptide, or an RNA molecule), such that the nucleic acid molecule that is positioned for expression in a cell is expressed in the cell.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription of a desired nucleic acid molecule. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent nucleic acid molecule expression controllable for cell type-specific, tissue-specific or inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene, and may be placed, by standard recombinant DNA manipulations, adjacent to or within the desired nucleic acid molecule.
By xe2x80x9coperably linkedxe2x80x9d is meant that a nucleic acid molecule and one or more regulatory sequences are connected in such a way as to permit expression of the nucleic acid molecule when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By xe2x80x9creporter genexe2x80x9d is meant any gene that encodes a product whose expression is detectable. A reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., luciferase or chloramphenicol acetyl transferase), toxicity (e.g., ricin), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labelled antibody).
By xe2x80x9cconserved regionxe2x80x9d is meant any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity between two or more of the p28 Bap31 family members, (e.g., between human p28 Bap31 and murine p28 Bap31).
By xe2x80x9cdetectably-labelledxe2x80x9d is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA or RNA molecule. Methods for detectably-labeling a molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32P or 35S) and nonradioactive labeling (e.g., chemiluminescent labeling, e.g., fluorescein labeling).
By xe2x80x9cantisense,xe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence that is complementary to the coding strand of a gene, preferably, a p28 Bap31 gene. Preferably the antisense nucleic acid molecule decreases the amount of transcription from the gene; more preferably, the decrease is at least 10%, and most preferably, the decrease is at least 50% when administered at the maximally effective dose.
By xe2x80x9cpharmaceutically acceptable carrierxe2x80x9d is meant a carrier that is physiologically acceptable to the treated mammal while retaining the therapeutic properties of the compound with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline solution. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington""s Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments.